VBNC, previously unrecognized in the life cycle of Porphyromonas gingivalis?

ABSTRACT Bacteria are exposed to stresses during their growth and multiplication in their ecological systems to which they respond in multiple ways as expert survivalists. One such response mechanism is to convert to a viable but not culturable (VBNC) state. As the name indicates, bacteria in the VBNC state have lost their ability to grow on routine growth medium. A large number of bacteria including many pathogenic species have been reported to be able to enter a VBNC state. VBNC differs from culturable cells in various physiological properties which may result in changes in chemical resistance, adhesion, cellular morphology, metabolism, gene expression, membrane and cell wall composition and/or virulence. The ability of VBNC bacteria to return to the culturable state or resuscitate, when the stressor is removed poses a considerable threat to public health. There have been few publications that overtly describe the ability of oral pathogenic species to enter the VBNC state. However, the presence of VBNCs among oral pathogens such as Porphyromonas gingivalis in human chronic infections may be an important virulence factor and have severe implications for therapy. In this review, we intend to i) define and summarize the significance of the VBNC state in general and ii) discuss the VBNC state of oral bacteria with regard to P. gingivalis. Future studies focused on this phenomenon of intraoral VBNC would provide novel molecular insights on the virulence and persistence of oral pathogens during chronic infections and identify potential novel therapies.


Introduction
Bacterial species have evolved multiple mechanisms to survive ecological, nutritional and chemical stresses. Among these is to enter a viable but not culturable (VBNC) state [1]. As the name indicates, bacteria in the VBNC state have lost the ability to grow on routine bacteriologic media [2]. Despite their inability to grow on usually permissive media, VBNC cells are, in fact, not dead nor spores, although the differences between VBNC and culturable cells are considerable. These include differences in chemical resistance, adhesion properties, cellular morphology, metabolism, gene expression, membrane and cell wall composition and virulence properties [2]. Yet, VBNC cells retain complete membranes, maintain the ability to synthesize mRNA, are metabolically active, carry out respiration, and take up and incorporate amino acids into proteins [2]. Researchers globally have identified about 100 species of bacteria that can enter the VBNC state. The VBNC state is also observed to be present in yeasts such as . At least one bacterial species has been reported to express a virulence factor during the VBNC state [3].
The stressful conditions that induce bacterial cells to enter the VBNC state are known for numerous species and have been found to vary not only among species but also strains. Some of the more common stressors described to date include starvation, antibiotic treatment and temperature changes [2]. Most importantly, VBNC bacteria have the ability to return to the culturable state or resuscitate, when the stressor is removed or negated. The length of time during which bacteria in the VBNC state can be resuscitated, the resuscitation window, varies widely among species. For example, it has been reported to be as short as 3 days for Vibrio vulnificus [4] or as long as 11 years for Citrobacter freundii [5].
Pathogenic VBNC bacteria initially gained attention due to their ability to exist at non-detectable thresholds by traditional water and food safety techniques. Several pathogens in this state may contribute initially to non-apparent infections, which are later followed by a manifestation of the disease. For example, Mycobacterium tuberculosis has been demonstrated to exist in a VBNC state and latent TB reactivation may be a attributed to resuscitation from the VBNC state [6]. Importantly, as reported by Oliver [7], the VBNC state of bacteria including Enterococcus faecalis, Escherichia coli, Haemophilus influenza, Helicobacter pylori and Mycobacterium smegmatis was significantly associated with increased resistance to antibiotics.

VBNC: a distinct physiological state of survival
The VBNC state was first discovered in 1982 in E. coli and Vibrio cholerae cell [1] and to date has been reported to exist in nearly 100 diverse bacterial species living in a wide range of environments [2,8,9]. The VBNC state represents a distinct physiological state. These bacteria can be identified by various detection methods to be alive and capable of subsequent resuscitation back to a culturable state. In general, the majority of bacterial species that have been observed to exist in the VBNC state are free living and exist in broad environmental conditions including food, water, and soil.
The VBNC state of bacteria was traditionally referred to as a dormant bacterial state. However, it is now clear that there exists a difference between VBNC and dormant stages based on their metabolic activity. The VBNC state is characterized by a metabolic activity that is always measurable, whereas during the dormant state, this activity declines to levels that are lower than the level of detection (Table 1). Thus the VBNC state is now widely accepted as a distinct physiological state of survival of bacteria [2,10]. This physiological state is not only a mechanism employed by bacteria to support their long-term survival during unfavorable conditions but may also contribute to cellular decay as a mechanism of preserving specific features of viable cells which results in an 'intentional' net loss of culturable characteristics.

Changes in cellular morphology during the VBNC state
Cellular morphology is a critical aspect of the phenotype of a cell. Since the cell wall/peptidoglycan determines the shape of the cell, as it enables the tensile strength and diffusion barriers that are necessary to attain a particular shape, any differences in the cell wall composition may affect the cellular morphology. This occurs in some VBNC bacterial cells as many have shown differences in cellular morphology such as cell dwarfing and rounding [11,12]. It is likely that a reduction in cell size is a strategy to limit the energy demands of VBNCs [13][14][15]. For example, Campylobacter spp. change from the characteristic spiral shape during normal exponential phase growth to a coccoid shape during transition to the VBNC state [16]. Similarly, Burkholderia pseudomallei and V. cholerae cells were also observed to change morphology from their characteristic rod to cocci shape during transition to the VBNC [17,18]. However, these morphological changes commonly seen in VBNC cells are not exclusive to VBNC cells, as similar changes are observed in non-VBNC cells that live under stressful conditions. Hence, a morphological change alone cannot be used to define the VBNC state [19].

Induction of the VBNC state
Bacterial cells undergo a change in their physiological state to VBNCs in response to various conditions including but not limited to environmentally unfavorable conditions [7], starvation of nutrients [12], temperature fluctuations [20], incubation outside optimal pH [21], changes in osmotic concentrations [22], differences in oxygen concentrations [23], heavy metal exposure [24,25], and importantly on exposure to antibiotics. Since there is a broad range of bacterial species that can enter the VBNC state, it is likely that diverse regulatory mechanisms control this state among these species. Some genes that are reportedly involved in regulation of the VBNC state in E. coli and Salmonella typhimurium include rpoS and oxyR. rpoS is a sigma factor that is essential for survival in stationary phase as well as during stress responses [26,27]. oxyR is a lysR-type transcriptional regulator which has a characteristic N-terminal DNA-binding domain that is known to regulate oxidative stressrelated genes and plays a crucial role in the induction of the VBNC state [28,29].
The lower metabolic rate of VBNCs results in changes in proteins, fatty acids and peptidoglycan in their cell walls and membranes. For example, there was an observed shift in the outer membrane subproteome in E.coli VBNCs including the levels of outer membrane proteins (Omp), the 43 β-subunit antigen, TolC, and OmpT while there was a shift in 106 proteins in VBNCs resulting from exposure to natural seawater and light [30]. Significantly, Omp W was found to be highly induced in VBNC cells [22]. During transition of V. vulnificus cells to the VBNC state, the levels and composition of unsaturated fatty acids increased with a major shift towards fatty acids with less than 16 carbons and increases in hexadecanoic, hexadecenoic, and octadecanoic acids [31]. Further, an increase in peptidoglycan cross-linking was also observed in E. faecalis VBNCs compared to culturable cells [32]. VBNC cells also have a variable gene expression profile compared to their culturable counterparts. For example, 58 genes related to regulatory functions, cellular processes, energy metabolism as well as transport and binding were all induced by more than 5fold in V. cholerae [33], while another study found a reduction in 16S rRNA as well as the mRNA levels of tuf, rpoS, and relA genes that are responsible for protein synthesis and stress responses [34].
These regulatory and physiological changes produce VBNCs that exhibit strong resistance to various physical, chemical and temperature stress conditions. For example, VBNC cells of V. vulnificus were observed to be resistant to mechanical destruction by sonication [35], while those of M. smegmatis were found to be more resistant to high temperatures [36]. VBNC cells of Vibrio parahaemolyticus [37], Campylobacter jejuni [38] and E. faecalis [39], were also found to show greater levels of resistance against low salinity, ethanol, and chlorine, respectively.

Detection of VBNC
An array of viability markers have been used to differentiate viable and VBNC cells. The most common method is differential staining combined with direct microscopic enumeration, the LIVE/ DEAD® Bac Light™ assay. This assay employs two fluorescent dyes, Syto 9 and propidium iodide, which have variable cell permeability characteristics that can differentiate cells with different membrane integrities [21].
The p-iodonitrotetrazolium violet (INT) assay, is another method and is based on the activity of an electron transport system [40]. As only viable cells can undergo metabolic activity and respiration, this assay can be used to differentiate the VBNC and dead cells [15]. INT is a soluble tetrazolium salt which competes with oxygen for electron acceptance and on reduction turns to an insoluble formazan complex in metabolically active cells. Hence, the formation and accumulation of formazan in cells, which is observed as a dark red precipitate under a microscope, indicates viable cells. The tetrazolium salt, 5cyano-2,3-ditolyltetrazolium chloride (CTC) or the BacLight™ RedoxSensor™ Green have also been used [20,41].
Untargeted metabolomics is being developed as a new method to identify the VBNC state [42,43]. With the increased metabolomics research capabilities, metabolomics technology is continuously evolving and has the potential to identify precise metabolic markers that could easily distinguish between culturable and VBNC cells based on the presence of metabolomic markers in the future [44]. Diphenyleneiodonium (DPI) treatment was used as an inducing agent to cause rapid transition in mycobacteria from an active state into a viable, but non-cultivable state, and comparing their characteristics with dormant phenotypes using untargeted metabolomics [43].

Resuscitation of VBNC cells to a culturable state
The term 'resuscitation' was first used to describe the recovery of non-culturable Salmonella enteritidis cells with the subsequent addition of HI broth. Two decades later, this phenomenon was again reported by Baffone et al. [45], who defined it as the state of reversal of metabolic and physiological changes that characterize VBNC cells. This phenomenon is hugely significant in the bacterial life cycle and poses a significant threat to human health. This was first realized for diseases caused by pathogens in the food or water supply since the safety of water and foods is routinely determined by plate counts of culturable cells. Consequently the presence of VBNC cells in a water or food sample results in spuriously low bacterial (or zero) plate counts which then is erroneously interpreted that the water/food is safe for consumption.
Another common threat, especially in the case of several host-associated human pathogens, occurs when an antibiotic is prescribed to treat an infection. The presence of an antibiotic can induce a bacterium to enter the VBNC state. In some cases, when the antibiotic is discontinued, the VBNC bacteria resuscitate, allowing the infection to reoccur. There is recent evidence that suggests some intracellular pathogens can enter the VBNC state inside their host cells, thus 'hiding' from the cell's antibacterial mechanisms and allowing them to persist. This ability of a bacterial species to enter a state of VBNC has been linked to the survival and pathogenicity of multiple host-associated species including Pseudomonas aeruginosa and M. tuberculosis [2,46]. For example, it is now recognized that the chronicity of tuberculosis is due to M. tuberculosis existing in a VBNC state.
The signaling mechanisms of resuscitation are not well understood because there are no readily available methods/techniques that can distinguish between culturable cells and those that arise from resuscitation. Thus far documented resuscitation has been reported only in approximately half of the human pathogens that are known to exist in the VBNC state. Thus mechanisms that govern resuscitation of pathogenic VBNC cells are mostly unknown which complicates more fully understanding of host-bacterial interactions and outcomes.
The issue becomes further complicated because of the 'resuscitation window', as first proposed by Pinto et al. [19] and defined as the time period during which VBNC cells retain their ability to resuscitate under normal suitable stimuli. It was observed that on exposure to inducing conditions, different cells in a population enter into a VBNC state at different times. Based on this, a hypothesis was proposed which stated that VBNC cells belonging to the same species will have a fixed resuscitation window, in that the older VBNC cells will lose their resuscitation ability earlier than the younger VBNC cells, resulting in a reduction of total resuscitable cells over time. Data from multiple studies have supported this hypothesis, as a reduction in the number of resuscitable cells over time was found in both V. cholerae and E. faecalis [18,47].
Various factors have been tested as a stimulus for resuscitation including a shift in temperature [48], the use of gas mixtures [49], the addition of amino acids [9], rich media [50], supernatant from spent culture medium [51][52][53], the addition of fresh host cells [54], and the addition of antioxidants to culture plates, among others [11,47,55]. It was also shown that the addition of specific compounds such as amino acids, resuscitation-promoting factors (Rpfs) and autoinducers [9,51,53] could resuscitate some species of VBNC cells.

The differences between persistence and VBNC states
The VBNC and persistence states were independently described decades apart but the phenotypic similarities between them require definitions that distinguish VBNC cells from persistent cells. Persistence occurs when the majority of a population of the cells are susceptible to an antibiotic, while a subpopulation of the cells are tolerant to the antibiotic. Thus persistent cells can be considered as phenotypic variants in a population [56]. Similar to persistent cells, VBNC cells also show antibiotic tolerance, and tolerance to various other stress conditions including exposure to heavy metals, high and low temperatures, fluctuation in pH, oxidative and osmotic challenges, and ethanol [57]. Hence, the line of distinction between 'persistence' and 'viable but non culturable' cells is quite thin. However, a crucial difference is their resuscitation dynamics. While persisting bacteria are characterized by a typically shorter resuscitation period and ability to grow on nutrient media immediately following the removal of antibiotics, VBNC cells require a longer resuscitation period after removal of the inducing stress. In addition, VBNC cells require an external stimulus in order to restore metabolic competence and to repair their damaged proteins that are necessary for their growth.
Bamford and colleagues, using a single-cell imaging technique, proposed a new identification and isolation system for persisters and VBNC cells which is based on their differential promoter activity expression [58]. This method has the potential to reveal a deeper understanding of the molecular changes that cells undergo during their transition from active growth to VBNC.

VBNC in the oral cavity?
The oral cavity can be defined as a mixture of distinct dynamic ecological habitats that support growth of a specific microbial community because of their characteristic biological features. To date, there are no publications that overtly describe the ability of P. gingivalis or any other oral species to enter the VBNC state, with the exception of E. fecalis for which the oral cavity is a secondary habitat. However, we propose such a state not only occurs but is key to the survival of P. gingivalis in vivo, thereby contributing to chronic infections.

P. gingivalis and VBNC
There are few publications that provide support for this hypothesis. First, laboratories worldwide attempted for many years to culture periodontal bacteria from diseased atherosclerotic vessels without success. This was partially in response to the criticism that studies that identified genomic DNA of oral pathogens in these diseased tissues did not prove the presence of viable pathogens, only the presence of the DNA, which could have been transported to the diseased site by macrophages. Then, in 2005, our laboratory published the first proof of the presence of viable P. gingivalis in diseased tissues [59]. This was accomplished by adding carotid atherosclerotic plaque homogenate directly to human cardiovascular aortic endothelial cells (HCAEC) and visualizing the presence of P. gingivalis within the ECV-304 cells using species-specific fluorescently tagged antibodies combined with deconvolution microscopy. This determination was crucial, as dead P. gingivalis cannot invade these nonphagocytic cells. Thus, blood agar did not contain the signal for growth and division but host cells did. The intriguing question then became, what was it about P. gingivalis in vivo that was different from P. gingivalis cultured in vitro?
A second important publication, by Li et al. [60], demonstrated that intracellular P. gingivalis strain W83 in either endothelial or smooth muscle cells could be enumerated by colony counting, for up to 48 hours of co-culture. However after 48 hours, the numbers of colonies detected on plates decreased to essentially zero. This occurred at the same time numerous intact P. gingivalis were visualized inside the cells by microscopy. Furthermore, when the infected cells were lysed and added to uninfected cells, there was a significant number of colonies culturable on blood agar plates. These results suggested that the intracellular P. gingivalis, although not culturable after 48 hours, were nevertheless viable and the addition of fresh uninfected host cells provided a signal for transformation into the culturable state.
Finally, Kozarov and coworkers were subsequently able to culture and isolate additional strains of P. gingivalis from atheroma plaques using the co-culture approach (Kozarov and personal communication). We have spent the ensuing years contemplating the state of P. gingivalis in human atheromatous tissues that prevents them from growing on normally permissive medium. Our hypothesis is that they become VBNCs while residing intracellularly in vivo.
P. gingivalis has been linked to a large number of other systemic chronic diseases, some of which include cardiovascular diseases, rheumatoid arthritis, preterm birth, Alzheimer's disease, and several types of cancers. In a recent observation by Ursula H et al., 2020, P. gingivalis was observed to exist in a VBNC state in neuronal cultures in vitro. We propose that the chronicity of P. gingivalis associated infections, both oral and systemic, is due to the ability of P. gingivalis to enter the VBNC state and subsequently periodically resuscitate within cells of the oral cavity or elsewhere within the body.
Most investigations of the VBNC state to date have studied bacteria in freeliving conditions, without the complication of the bacterial cells being intracellular. However, it was recently reported that intracellular Listeria monocytogenes changes from the cytoplasmic motile lifestyle, in which it uses host cell actin to transit from one host cell to another, to a state in which the bacteria lose their ability to interact with actin and instead localize within LAMP1 positive lysosomal-like vacuoles [61]. Surprisingly, the bacteria localized to lysosomes are able to resist degradation and enter into a nonreplicative state, determined to be VBNC. The authors also reported that L. monocytogenes can 'revert' to its normal state of replication upon co-culturing with the infected host cells. The authors suggest that VBNC represents a state of persistence that can then result in the asymptomatic carriage of this bacterium by human hosts, thereby lengthening the incubation period of listeriosis and promoting L. monocytogenes survival/resistance.

Evidence of the VBNC state and P. gingivalis
Based on the studies cited above, we began an investigation of the possibility of P. gingivalis entering into a VBNC state. Our first experiments were limited to in vitro conditions. We reasoned that oxidative stress would be one condition to investigate, since P. gingivalis encounters oxidative stress in vivo. To this end, oxidative stress was induced by the addition of 10 mM hydrogen peroxide to stationary P. gingivalis cultures. After 30 min of exposure, the cultures were plated to determine colony-forming units (CFUs) and viability was assayed using the LIVE/DEAD BacLight Bacterial Viability Kit. As can be seen in Figure 1(a), no CFUs were detected on blood agar plates. However, LIVE/DEAD backlight staining demonstrated the presence of numerous viable bacteria (Figure 1(b)), indicating that P. gingivalis strain W83 had indeed entered the VBNC state after exposure to oxidative stress.
We next attempted resuscitation of the VBNC cells that were exposed to H 2 O 2 by the addition of 0.6 mM sodium pyruvate for 60 min. The rationale behind the selection of sodium pyruvate was due to the cytoprotection ability of it against oxidative stress. These cultures were then plated and viability was determined. As can be seen in Figure 2, we observed that on exposure to sodium pyruvate the VBNC bacteria became culturable and viable, indicating that W83 was resuscitated from the VBNC state by the addition of sodium pyruvate.
Subsequently, we investigated the possibility that intracellular P. gingivalis strain W83 could convert to the VBNC state. Using our human Coronary Artery Endothelial Cell (HCAEC) model, we observed that after 72 hours of infection, no viable P. gingivalis could be detected by plate counting (Figure 3(a)). However, again using live/dead staining, we Figure 2.Resuscitation of PgW83 in the VBNC state to the culturable state. Following H2O2 treatment, PgW83 was no longer culturable but viable according to Syto9 staining indicating PgW83 was in the VBNC state (see Figure 1). When treated with sodium pyruvate following H2O2 stress, PgW83 was viable (Syto9 staining not shown) and culturable on blood agar plates indicating the cells have been resuscitated. determined that the intracellular bacteria were indeed alive (Figure 3(b)). Together, these data suggested that P. gingivalis strain W83 can exist in a VBNC state not only under in vitro conditions but also in intracellular conditions. Also, when the VBNC inducing condition is removed, P. gingivalis W83 can resuscitate.
Furthermore, we have identified a number of putative PgW83 homologues of gene products that have been used as VBNC markers or required for the VBNC or resuscitation states in other bacteria (Table 2). PorR, Pg1393, and Pg0575 show homology to the VBNC markers in E. coli and E. faecalis. RpoD in PgW83 is a putative homologue of rpoS, an RNA polymerase sigma factor, required for E. coli to enter the VBNC state. PgW83 has a comparable two-component signaling system (Pg1797 and Pg1089), which has been proposed to be a sensor to initiate the VBNC state in E. coli. PgW83 also has homologues to two Vibrio genes required for the VBNC state. Finally, we have identified possible PgW83 homologues to resuscitation genes characterized in Rhodococcus, Mycobacterium, and Vibrio.
These findings offer new insights into the lifecycle of P. gingivalis strain W83 and likely other strains of P. gingivalis as well. We now are aware that P. gingivalis strain W83 appears to have the necessary genes for the VBNC state and resuscitation and is capable of entering a VBNC state and can resuscitate, once an appropriate environment/signal is present, to a culturable state. These observations are significant not only in terms of the pathobiology of P. gingivalis strain W83 but also call into question the conclusions of previously published studies of trafficking of P. gingivalis within host cells.
The previously published investigations should be reevaluated and reinterpreted, since the vast majority of such studies used only CFU enumeration to determine live bacteria within the host cells.

Other oral bacteria
It is almost certain that other oral bacterial species can enter and resuscitate from a VBNC state. Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguinus were also reported to display phenotypes that closely resemble the description of VBNC states. E. fecalis for which the oral cavity is a secondary habitat is observed to adopt the VBNC state in response to environmental conditions. As additional studies are done, we expect that a variety of oral bacteria will also demonstrate that the VBNC state is part of their lifestyle. Thus investigation of the VBNC state in oral bacterial pathogens is vital to more completely define and characterize all phases of their lifestyle, thereby more fully understanding their virulence/survival mechanisms. This understanding of a previously unknown lifestyle should result in new and novel preventative approaches and treatments for oral and other diseases. For example, the identification of the molecular determinants required for the transition of P. gingivalis to the VBNC state and/or resuscitation during infection could lead to the development of therapeutics that target the chronicity of infections by this bacterium. In addition to antimicrobials, disinfectants also have been shown to trigger entry into the VBNC state. It is thus entirely possible that disinfecting agents used in oral care, e.g. in endodontics may induce the associated oral bacteria to enter the VBNC state. The chronic presence of VBNC of oral bacteria may have severe implications for therapy for infections of the oral cavity as well as infections caused by these pathogens at various and multiple sites within the body.

Future perspectives
The precise role of the VBNC state in bacterial pathogenesis is yet to be elucidated. It is possible that its role and significance differ from bacterium to bacterium. It will be crucial to understand the role of VBNCs in evasion of host immune detection. By this publication, we seek to convey to the community studying the oral microbial populations that there is a need to initiate studies to further investigate the significance, role and molecular signaling mechanisms of the VBNC and resuscitation states not only in P. gingivalis but other pathogens as well. More importantly, future studies of this previously unknown and unappreciated stage of the life cycle of oral pathogens will define mechanisms that can be targeted for novel antimicrobial agents designed to interfere with the VBNC and/or resuscitation states. The identification of such drug targets would have the potential to prevent the persistence and chronicity of not only P. gingivalis infections, but those of other bacterial species involved in chronic diseases.